Serum levels of leptin, ghrelin putative peptide YY-3 in patients with fetal alcohol spectrum disorders

Fetal alcohol spectrum disorders (FASD) are a severe developmental condition resulting from exposure to alcohol during pregnancy. The aim of this study was to examine the concentrations of hormones involved in appetite regulation—ghrelin, leptin, and putative peptide YY-3 (PYY)—in the serum of individuals with FASD. Additionally, we investigated the relationship between these hormone levels and clinical indicators. We conducted an enzyme-linked immunosorbent assay on samples collected from 62 FASD patients and 23 individuals without the condition. Our results revealed a significant decrease in leptin levels among FASD patients compared to the control group (5.124 vs. 6.838 ng/mL, p = 0.002). We revealed no statistically significant differences in the levels of other hormones studied (ghrelin and PYY). Comparisons of hormone levels were also conducted in three subgroups: FAS, neurobehavioral disorders associated with prenatal alcohol exposure and FASD risk, as well as by sex. Assignment to FASD subgroups indicated changes only for leptin. Sex had no effect on the levels of hormones. Moreover, the levels of leptin showed a negative correlation with cortisol levels and a positive correlation with BMI and proopiomelanocortin. Alterations in appetite regulation can contribute to the improper development of children with FASD, which might be another factor that should be taken into consideration in the proper treatment of patients.


Study group
A single-center cross-sectional study was carried out in 62 patients with FASD aged 5 months to 16.5 years and 23 healthy controls.The affected individuals were categorized into three subgroups: FAS, ND-PAE (neurobehavioral disorder associated with prenatal alcohol exposure) and FASD risk.Diagnosis of FASD was based on the most recent Polish recommendations 24 .In respect of internationally adopted guidelines, the ND-PAE domain includes: partial fetal alcohol syndrome, and alcohol-related neurodevelopmental disorder [4][5][6][7] .Participants were included from the Department of Pediatrics, Pediatric Endocrinology and Diabetology and the Endocrinology Outpatient Clinic between March 2019 and January 2022.All procedures involving human participants adhered to the ethical standards of the institutional and/or national research committee and were in line with the Declaration of Helsinki of 1964 and its subsequent amendments or comparable ethical standards.Informed consent was obtained from all participants or, for those under 16 years of age, from a parent and/or legal guardian.The study protocol received approval from the Institutional Ethics Committee of the University of Rzeszow (16/02/2019).

Blood sampling
Blood sampling was conducted in the morning between 8:00 and 10:00 am, following an overnight fast.Subsequently, the blood was incubated at room temperature for at least 30 min but no more than 2 h, and then centrifuged (1500 × g, 10 min, 4 °C).The resulting serum was transferred to cryovials and stored at − 80 °C in a freezer until further analysis.

Hormone levels determination: leptin, ghrelin, and putative peptide YY
The serum hormone levels were assessed in duplicate following prior dilution using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Fine Biotech Co., Ltd., Wuhan, China), as per the manufacturer's instructions.The limit of detection for each hormone was determined as follows: leptin, 18.75 pg/ ml; ghrelin, 1.125 pg/ml; putative peptide YY-3, 3.75 pg/ml.The within-assay and between-assay coefficients of variation were both found to be below 8% and 10%, respectively.Clinical parameters were retrieved from patient medical records, while blood morphology was analyzed using a hematology analyzer from Siemens Healthineers, Germany.

Statistical analysis
All statistical analyses were conducted using the STATISTICA software package (version 13.3, StatSoft Inc. 2017, Tulsa, OK, USA).The data were presented as either mean and standard deviation or median with the range.Most variables did not adhere to a normal distribution, as confirmed by the Shapiro-Wilk test, necessitating the utilization of non-parametric tests.For comparisons between two independent groups, the Mann-Whitney U test was employed, while for multiple comparisons, the Kruskal-Wallis ANOVA was utilized.Correlation analysis was performed using the Spearman correlation test.A p-value of less than 0.05 was considered statistically significant.

Results
The study included 62 patients with FASDs, comprising 31 boys and 31 girls.Simultaneously, 23 healthy children, consisting of 16 boys and 7 girls (30.5%), were enrolled in the study.Table 1 presents the basic anthropometric and clinical laboratory parameters of both FASD patients and healthy controls.
With the exception of BMI percentile, none of the parameters under investigation exhibited statistically significant distinctions.There were no notable variations in age between patients diagnosed with FASD and those in the healthy control cohort.However, we did observe a significant age disparity within the FASD subgroups (refer to Table 2), largely due to the younger age of individuals in the FASD risk category.Nonetheless, the disparities between the FAS and ND-PAE groups did not reach statistical significance (p = 0.686).As mentioned above, the BMI percentile values were markedly lower in the FASD cohort compared to the control group (p = 0.035).Differences in BMI were associated with disease severity and the BMI percentile in the FAS subgroup was notably lower than that in the ND-PAE group.
In the FAS subgroup, 42.31% of children exhibited low height (< 3rd percentile), with the majority being girls (90.91%) compared to boys (9.09%).Conversely, within the ND-PAE subgroup, 18.18% of children had low height, with the largest segment falling between the 25th and 50th percentiles (27.27%), evenly distributed between girls and boys (50% each).In the FASD RISK group, 20% of children displayed low growth.No statistically significant differences were noted between the groups in terms of biochemical parameters such as lipid profile, glucose, insulin, or HOMA-IR.
The hormone levels examined are illustrated in Fig. 1.A notable reduction in leptin levels was observed in the serum of individuals with FASD compared to healthy counterparts (p = 0.002).Significant differences in leptin levels were also observed (Table 3).However, caution is warranted in interpreting these results due to the potential confounding effect of younger age observed in FASD risk patients, which may impact leptin secretion.Specifically comparing the FAS and ND-PAE groups revealed no disparities (p = 0.732).Concentrations of other studied hormones, such as PYY and ghrelin, demonstrated similarity between individuals with FASD and controls, as well as within FASD subgroups.Table 4 provides a comparison of hormone concentrations between females and males with FASD, indicating no discernible differences in leptin, ghrelin, and PYY levels between sexes in the FASD cohort.
The next stage of data analysis involved evaluating the correlations between the hormones levels of the studied and the clinical parameters of patients with FASD (Table 5).Leptin showed a positive correlation with blood glucose concentration (r s = 0.289, p = 0.028) and a negative correlation with cortisol levels (r s = − 0.307, p = 0.018).Putative peptide YY-3 and ghrelin were not correlated with any clinical parameter.Furthermore, we examined the correlations between the hormones tested and other appetite-regulating hormones, including proopiomelanocortin (POMC), Agouti-signaling protein (ASP), kisspeptin (KISS1), neuropeptide Y (NPY) and alpha-melanocyte-stimulating hormone (α-MSH), which we previously determined by us 25,26 .We found weak correlation with POMC: a negative for PYY (r s = − 0.289, p = 0.024) and positive for ghrelin (r s = 0.268, p = 0.042).Leptin was also positively associated with the levels of POMC (r s = 0.339, p = 0.007) and ASP (r s = 0.31, p = 0.014).
Table 1.Baseline demographic and clinical data of the study participants.BMI percentile: body mass index percentile; LDL: low-density lipoprotein; HDL: high-density lipoprotein; HbA1c: glycated hemoglobin; HOMA-IR: homeostasis model assessment of insulin resistance, data are presented as median and range; differences between means were analyzed using Mann-Whitney U test.Significant values are in bold.

Discussion
This study has investigated the effects of prenatal exposure to ethanol on serum levels of hormones that regulate appetite: leptin, ghrelin and putative peptide YY-3.The main finding of our study is a significantly decreased leptin level in FASD compered to healthy control.We have also observed correlations of leptin levels with glucose, POMC, ASP and cortisol.No significant differences were found in PYY and ghrelin concentrations between FASD subgroups as well as controls.Sex did not affect the levels of hormones studied.Leptin, an anorexigenic hormone discovered in 1994, is primarily secreted from white adipose tissue.It acts through the activation of transmembrane leptin receptors, which are widely distributed in peripheral organs as well as the central nervous system 27 .Feeding or obesity increases circulating leptin levels, while fasting decreases them 28 .Besides regulating food intake and body mass, this hormone also influences reproductive functioning and plays a vital role in fetal growth, proinflammatory immune responses, lipolysis, and angiogenesis 21,29,30 .Its expression and secretion are regulated by many factors, including inflammatory cytokines, insulin, and glucocorticoids 31 .Recent studies have underlined the impact of alcohol consumption on increased short-term energy intake due to appetite stimulation, leading to weight gain and obesity 32,33 .
Alcohol exerts an inhibitory effect on leptin secretion, although the data are not consistent 34 .Alcohol can also affect the leptin receptor.In mice consuming ethanol for 5 weeks, the overall expression of leptin receptors was elevated, whereas the expression of the functionally active long form of leptin receptor decreased in the hypothalamus and perigonadal fat 35 .In humans, soluble leptin receptor levels in alcohol-dependent patients were higher than in the control group and decreased during the abstinence program 36 .Based on these studies, it can be assumed that improper development in children with FASD may be associated with alterations in leptin receptor expression.Furthermore, leptin indirectly regulates the hypothalamic-pituitary-adrenal axis and the cortisol-mediated stress response, possibly influencing alcohol craving and withdrawal 37 .There is growing evidence that appetite-regulating peptides such as leptin and ghrelin are altered in alcoholism.The brain POMC system plays critical roles not only in regulating energy homeostasis but also in modulating alcohol intake 38 .The POMC system, which is susceptible to ethanol influence, is also intricately linked with leptin regulation.Ethanol exposure harms POMC neurons and decreases its secretion 25,39,40 .Furthermore, some evidence indicates that leptin can regulate human adrenal function through its receptors in the hypothalamus, where it modulates the secretion of corticotropin-releasing hormone and, consequently, ACTH in the pituitary 41 .Leptin inhibits ACTH-stimulated steroid release by all three zones within the adrenal glands, with the most severe reduction in cortisol production 42 .This may be a protective mechanism opposite to the local situation in visceral adipose www.nature.com/scientificreports/tissue, where glucocorticoid receptors and 11β-hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to active cortisol, become upregulated in obesity 43 .These findings are supported in our study, by the results of correlation analysis, which reveal negative correlations between leptin and cortisol levels, as well as positive correlations between leptin and POMC.However, contrary to many previous studies, we did not find any association between the concentration of circulating leptin and the BMI of patients 21,[44][45][46] .Although, as in our study, several reports also found no correlation between BMI and leptin levels 47 .Higher serum leptin levels www.nature.com/scientificreports/have been shown to correlate with lower BMI in childhood and a reduced predisposition to developing metabolic disorders in adolescence and adulthood 48 .The effect of leptin on glucose metabolism and insulin secretion has been the subject of numerous studies 49,50 .We have found a weak positive correlation between leptin and glucose levels, but we have not observed any relationship between leptin concentrations and insulin, as well as insulin resistance indicators such as HOMA-IR, as previously described.Similar to previous studies, we found higher insulin concentrations in girls compared to boys, although the difference was not statistically significant 51,52 .
To date, there is a lack of knowledge about the effect of alcohol consumption during pregnancy on the secretion of appetite-regulating hormones, such as leptin or ghrelin, during infancy and childhood.Only a limited number of studies have addressed this issue.We have found significant differences in leptin levels among subjects with FASD compared to controls, as well as among FASD subgroups.The disease severity appears to negatively impact leptin secretion, except for the FASD risk group, which could be attributed to the significantly younger age of the participants.Children exposed to alcohol prenatally may display pre-and postnatal growth retardation, a defining characteristic of FASD 53 .Lower serum leptin levels were observed during the first 2 years of life in children exposed to ethanol 54 , which was also confirmed in our FASD risk subgroup, with median age 2.25 ± 1.26.Similar results were observed in animal model, where prenatal alcohol exposure led to reduced leptin levels in the subcutaneous adipose tissue of neonatal rats but not in adult rats 55 .
Ghrelin, the "hunger hormone," primarily originates from enteroendocrine cells in the stomach and regulates food intake by signaling hunger during fasting.It exhibits antagonistic action to leptin and its levels decrease in obesity due to positive energy balance [56][57][58] .Besides its presence in the arcuate nucleus of the hypothalamus, ghrelin receptors in the mesolimbic dopaminergic system, suggest its role in regulating rewards in substance use disorders such as alcohol addiction, primarily through activating the cholinergic-dopaminergic reward link 59 .Animal studies have shown the beneficial effects of ghrelin receptor antagonists in reducing the intake of palatable foods, suppressing preferences for caloric foods, and decreasing alcohol intake, as well as suppressing alcohol-induced reward 60 .Additionally, ghrelin demonstrates potent central gastroprotective activity against ethanol-induced lesions in animal models 61 .While the specific role of ghrelin in FASD remains unclear, studies in rodents suggest that prenatal alcohol exposure can alter ghrelin levels in offspring, potentially affecting appetite regulation and increasing the risk of obesity later in life [62][63][64] .We found a positive correlation between ghrelin and POMC, involved in energy balance and neuroendocrine function 65 .Early exposure to alcohol during pregnancy has been shown to alter the activity of the hypothalamic-pituitary-adrenal axis, increasing the risk of anxiety, depression, and alcohol-related disorders 25,66,67 .Contrary to previous findings, we did not observe a relationship between ghrelin levels and BMI or glucose levels 46,68 .The correlation analysis between hormones regulating appetite also indicated no association between leptin and ghrelin, which differs with many previous studies 57,69 .Our results showed no change in circulating ghrelin levels between the study groups; therefore, we can assume that the ghrelin mediation system is not disturbed by prenatal alcohol exposure.
Putative peptide YY-3, a gut-derived hormone, is recognized for its role in reducing short-term food intake by stimulating hypothalamic neuropeptide Y receptors (56).PYY is released from enteroendocrine cells, primarily found in the distal gastrointestinal tract postprandially in proportion to the ingested calories, reaching its maximum levels after 1-2 h 70 .The anorexigenic effect is likely induced by stimulation of the Y2 receptor in the arcuate nucleus, as feeding is abrogated in Y2 receptor knockout mice 71 .PYY slows gastric emptying and gastrointestinal motility, inhibits gastric acid secretion, gallbladder contractions, and secretion of pancreatic exocrine enzymes 72 .Several studies have investigated the association of PYY secretion with various clinical and nutritional parameters such as age, sex, BMI, adiposity, dietary fats, or acute exercise [72][73][74][75] .We did not find significant changes in PYY levels between participants with FASD and healthy individuals, nor within subgroups of FASD.The influence of fetal alcohol exposure on PYY secretion remains uncharacterized.Higher plasma levels of PYY have been reported in patients with anorexia nervosa compared to obese individuals, or morbidly obese as well as lean individuals 76 but no differences were also reported 77 .Significantly lower PYY concentrations have been described in children born small for gestational age, a common clinical features of FAS 78 .The effect of alcohol consumption was investigated by Calissendorff and colleagues 79 .They reported that unlike ghrelin, alcohol consumption does not affect changes in PYY levels.PYY levels have shown no significant changes after acute and chronic ethanol administration in animal models 80 .However, previous study has indicated stimulatory effect of PYY on POMC neurons 81 , which are damaged in fetus by prenatal alcohol exposure 40 .Thus, assessing the definitive effect of this exposure on nutrition regulation is challenging.Correlation analysis revealed only week negative association of PYY with POMC, and very close to statistically significant positive correlation with α-MSH (R = 0.250, p = 0.054).As mentioned before, previous studies have reported stimulatory effect of PYY on POMC expression and inhibitory effect on NPY/AgRP neurons, but these findings were not supported in our study 81 .In the correlation analysis of many appetite-regulating hormones, only a few were related, such as leptin and POMC, ASP and cortisol, ghrelin and POMC, PYY and POMC, which may suggest that food intake and hunger regulation is a complex and multifactorial process in which there is no direct activation/secretion of one hormone after the release of another, even if they act in opposing ways.
Although our study offers new insights into the hormonal regulation of food intake and energy balance in children prenatally exposed to alcohol, we acknowledge several limitations.The number of participants may not have been sufficient to definitively determine the effect of prenatal alcohol exposure on the subsequent secretion of leptin, ghrelin and PYY.The size of the subgroups, especially the FASD risk group, was relatively low, which could have affected the reliability of the statistical analysis.We also did not assess the intensity of physical activity in the study population, although it is known that exercise can alter the levels of ghrelin and PYY.Finally, the limited number of scientific studies describing the hormonal regulation of food intake in FASD complicates the interpretation of the results.

Conclusions
We found lower serum levels of leptin in patients with FASD compared to healthy subjects, while there was no difference in the concentration of ghrelin and PYY.The levels of hormones studied were not affected by sex or membership in FASD subgroups, except for leptin.We also observed some interesting correlations between the levels of the hormones studied and the parameters describing the clinical condition of the patients, including the association of leptin levels with glucose, POMC, ASP, and cortisol, as well as ghrelin and PYY with POMC.Basic anthropometric and clinical parameters such as age, BMI, or lipid profile did not correlate with any of the investigated hormones.However, the observed alterations in the rate of development among individuals with FASD may stem from disrupted appetite control.These findings should be considered in the appropriate and holistic management of patients.

Figure 1 .
Figure 1.Level of Leptin, Ghrelin, and Putative peptide YY-3 in patients with FASD as compared to healthy participants.

Table 2 .
Baseline demographic and clinical data of the FASD subgroups.BMI percentile: body mass index percentile; LDL: low-density lipoprotein; HDL: high-density lipoprotein; HbA1c: glycated hemoglobin; HOMA-IR: homeostasis model assessment of insulin resistance, data are presented as median and range; comparison between means were analyzed using Kruskal-Wallis test or "*"Mann-Whitney U test (comparison between FAS and ND-PAE only).Significant values are in bold.

Table 3 .
Leptin, ghrelin and putative peptide YY-3 levels in subgroups of patients with FASD.Data are presented as median and range; comparison between means were analyzed using Kruskal-Wallis test.Significant values are in bold.

Table 4 .
Hormone levels by sex of FASD patients.Data are presented as median and range; comparison between means were analyzed by Mann-Whitney U test.